Molecular identification method for single dinoflagellate cyst

Abstract

The present invention provides a molecular identification method for a single dinoflagellate cyst, including: obtaining crushed liquids of the single dinoflagellate cyst; performing a first PCR amplification with the crushed liquids as a template, SEQ ID NO: 1 (GTCCGCCCTCTGGGTG) as a forward primer, and SEQ ID NO: 2 (TCGCAGTAGTSYGTCTTTAAC) as a reverse primer to obtain an amplification product so as to determine the species of the dinoflagellate cyst. If the first amplification product is unsuitable for sequencing due to weak bands by electrophoresis detection, a second (nested) PCR amplification and sequencing is performed with SEQ ID NO: 1 (GTCCGCCCTCTGGGTG) as a forward primer and SEQ ID NO: 2 (TCGCAGTAGTSYGTCTTTAAC) as a reverse primer to determine the species of dinoflagellate.

What is claimed is: 
     
       1. A molecular identification method for a single dinoflagellate cyst, comprising:
 (a) dissolving a sample of a marine sediment in filtered seawater or artificial seawater to obtain a sample solution and performing ultrasonic treatment on the sample solution; 
 filtering the sample solution through a nylon mesh after ultrasonic treatment, intercepting particulate matter, and performing suspension treatment on the intercepted particulate matter to obtain liquids containing suspended particle matter; 
 looking for a single dinoflagellate cyst from the liquids containing suspended particle matter through a microscope, and extracting and transferring a liquid containing a single dinoflagellate cyst into a PCR tube; 
 cleaning the liquid containing the single dinoflagellate cyst using ultrasonic treatment, and obtaining a crushed liquid of the single dinoflagellate cyst; 
 (b) performing a first polymerase chain reaction (PCR) with the crushed liquid of the single dinoflagellate cyst as a template, SEQ ID NO: 1 (GTCCGCCCTCTGGGTG) as a forward primer and SEQ ID NO: 2 (TCGCAGTAGTSYGTCTTTAAC) as a reverse primer according to the following procedure: incubation at 95° C. for 5 minutes; cycling 30 times with successive cycles of 95° C. for 30 seconds, 57° C. for 45 seconds and 72° C. for 60 seconds; and elongation at 72° C. for 7 minutes, so as to obtain a first amplification product; 
 (c) checking whether the first amplification product is a target sequence using agarose gel electrophoresis; 
 (d) if DNA bands obtained in the agarose gel electrophoresis of step (c) show that the first amplification product meets a predetermined requirement, sequencing the first amplification product to determine the species of the single dinoflagellate cyst; and 
 (e) if DNA bands obtained in the agarose gel electrophoresis of step (c) show that the first amplification product fails to meet the predetermined requirement, performing a second PCR with the first amplification product as a template, SEQ ID NO: 1 (GTCCGCCCTCTGGGTG) as a forward primer and SEQ ID NO: 2 (TCGCAGTAGTSYGTCTTTAAC) as a reverse primer according to the following procedure: incubation at 95° C. for 5 minutes; cycling 30 times with successive cycles of 95° C. for 30 seconds, 57° C. for 45 seconds and 72° C. for 60 seconds; and elongation at 72° C. for 7 minutes, so as to obtain a second amplification product; and sequencing the second amplification product to determine the species of the single dinoflagellate cyst. 
 
     
     
       2. The molecular identification method of  claim 1 , wherein the first PCR comprises the following compositions: 10 μL of the crushed liquid of the single dinoflagellate cyst, 2.5 μL of a buffer solution, 2.0 μL of Mg 2+ , 20 ng of the forward primer, 20 ng of the reverse primer, and 1.5 U Taq polymerase. 
     
     
       3. The molecular identification method of  claim 1 , wherein in step (a), the nylon mesh has a pore size of 20 μm.